The technique of amplifying DNA using the polymerase chain reaction (PCR) assay is a powerful method for detecting low levels of DNA. We developed a simple and rapid PCR method for the detection of hepatitis B virus (HBV) DNA in serum. In testing serial samples from five chimpanzees experimentally infected with HBV, we found that HBV DNA was detected 2-3 weeks before the appearance of hepatitis B surface antigen (HBsAg), and continued to be detectable 1-3 weeks after the production of antibody to HBsAg. Sera from 98 patients at various stages of chronic HBV infection were also studied. Patients were divided into three groups according to their HBsAg and hepatitis Be antigen (HBeAg) status. Group I patients were positive for both HBsAg and HBeAg in the serum. PCR analysis demonstrated that all 31 (100%) of these patients possessed serum HBV DNA. Group II patients were positive for HBsAg and negative for HBeAg. Analysis showed that 36 of 46 (78%) of these patients possessed serum HBV DNA. Group III patients were former chronic HBV carriers but subsequently lost HBsAg during follow up. In this group 5 of 21 (24%) were found to harbor HBV DNA sequences in the serum. Detection of HBV DNA by PCR analysis is a valuable method for detecting the presence of DNA-containing virions in serum.